Recent accomplishments related to our therapeutic cancer vaccine clinical trials include the following: [] We have continued our preclinical and clinical investigations of two diverse recombinant (recombinant) vaccine platforms (each with demonstrated unique properties): (a) recombinant poxviral vectors employing a recombinant vaccinia prime followed by multiple rec. fowlpox booster vaccinations; each vector contains the transgenes for one or more tumor-associated antigens (TAAs) and three T-cell costimulatory molecules (designated TRICOM); and (b) heat-killed recombinant Saccharomyces cerevisiae (yeast) containing TAA protein via the insertion of a yeast plasmid. [] Prior Phase II studies with rV-, rF-PSA-TRICOM (PROSTVAC) vaccine as a monotherapy in patients with metastatic castration-resistant prostate cancer (mCRPC) have led to an ongoing Phase III study. This is a global 3-arm study in patients (n=1,200) with asymptomatic mCRPC who receive (a) PROSTVAC, (b) PROSTVAC + GM-CSF, (c) placebo (empty vector). The endpoint is overall survival (OS); accrual is expected to be completed in late 2014 or early 2015. [] We have evaluated the growth rates of tumors (via serum prostate-specific antigen (PSA)) in patients with mCRPC receiving PROSTVAC vaccine or several different chemotherapy regimens. These studies have revealed that, unlike chemotherapy, vaccine therapy can reduce the tumor growth rate leading to enhanced survival even in the absence of increases in progressive-free survival. All patients in this trial were evaluated for antibodies to PSA post-vaccination and were negative. Moreover, serum PSA values were in concordance with serum PAP values. [] Preclinical LTIB studies showed the superiority of combining intratumoral (i.t.) and systemic (s.c.) vaccination vs. the use of either alone. We have now completed a safety and feasibility study of combined i.t./s.c. PROSTVAC vaccination in men with locally recurrent or progressive prostate cancer. 19/21 patients developed stable or improved PSA levels. A follow-up trial has been initiated to evaluate post- vs. pre-vaccination prostate biopsies following s.c. vaccination only. [] Following LTIB preclinical studies, we have completed a Phase I trial in patients with mCRPC of PROSTVAC vaccination with increasing doses of ipilimumab (anti-CTLA4). No adverse events beyond those seen with ipilimumab alone were observed. The OS of patients was quite favorable compared with a similar mCRPC population receiving PROSTVAC alone in a separate trial. Studies by others in patients with metastatic prostate cancer have shown only limited improvement in OS employing ipilimumab alone. These findings provide evidence for the use of vaccine in combination with checkpoint inhibitors such as anti-PDL1. [] Two randomized studies have recently been completed employing PROSTVAC with standard-of-care therapies. Patients with non-metastatic CRPC received the testosterone-suppressing agent flutamide +/- PROSTVAC. This study provided the rationale for the ongoing trials with enzalutamide plus vaccine. In a second recently accrued trial, patients with mCRPC with bone metastases were randomized to receive the bone-seeking radionuclide Quadramet (Sm-153) +/- PROSTVAC. [] A dual center (NCI and MD Anderson) study randomized patients with metastatic breast cancer to docetaxel +/- PANVAC vaccine (rV-, rF-CEA-MUC1-TRICOM). Time to progression (TTP) in the docetaxel arm was 120 days vs. 192 days in the combination arm. The study revealed a striking decrease in TTP for patients who had received chemotherapy within 3 months of going on trial. [] We have completed a first-in-human Phase I study of recombinant yeast-CEA vaccine in patients with metastatic disease and demonstrated safety to the generation of CEA-specific T-cell responses. This has led to the initiation of a Phase II study in patients with metastatic medullary thyroid cancer (MTC), where the primary endpoint is tumor growth rate following serum calcitonin, which is a marker for disease progression. [] Two randomized clinical studies have recently been initiated employing the novel FDA-approved androgen blockade agent enzalutamide +/- PROSTVAC vaccine. The first is in mCRPC patients with a TTP endpoint; the second trial is in non-metastatic patients with rising PSA where changes in PSA velocity at the discontinuation of enzalutamide will be evaluated. [] A clinical study has opened in collaboration with Dr. P. Agarwal in the CCR Urologic Oncology Branch (UOB). Bacillus Calmette-Guerin (BCG) failure bladder cancer patients will be randomized to second-line BCG +/- PANVAC vaccine. The primary endpoint will be tumor extent and immune infiltrate in pre- vs. post-treatment biopsies. [] We have developed assays to analyze numerous soluble factors in sera pre- and post-immunotherapy. In collaboration with Dr. J. Gildersleeve in the CCR, we have profiled anti-glycan antibodies using glycan arrays in several of our PROSTVAC trials. Pre-vaccination levels of Abs to one specific glycan highly significantly (P=0.005) correlated with OS, while no correlations were seen in patients receiving empty vector placebo. Production of antibodies to a second glycan post- vs. pre-vaccination also correlated with OS. [] We have analyzed post- vs. pre-serum samples for a range of cytokines as well as two potential immunomodulatory molecules. We have shown that soluble CD40L (sCD40L) is elevated in the sera of patients with prostate cancer and metastatic breast and lung cancers. In vitro studies demonstrated sCD40L could enrich myeloid derived suppressor cells (MDSC) and expand Tregs. [] We have shown that soluble CD27 (sCD27) sera levels are decreased in carcinoma patients vs. healthy donors. mCRPC patients treated with PROSTVAC + ipilimumab showed a significant increase (P0.0005) in sCD27 post-vaccination, which was associated with increased OS (P=0.022). In vitro studies demonstrated that sCD27 provides strong proliferative signals to lymphocytes. [] We have now developed a multi-laser/multi-color FACS-based assay to analyze up to 50 different immune cell subsets in PBMCs pre- vs. post-treatment. In one example, there were associations with OS (P0.005) of several immune cell subsets pre-treatment with PROSTVAC + ipilimumab. [] We have used the multi-color FACS-based assay to define a peripheral immunoscore monitoring immune cell subsets known in the literature to have specific immune stimulatory or regulatory functions. In the randomized trial of metastatic breast cancer patients receiving docetaxel +/- PANVAC vaccine, there was no association of this immunoscore with increased PFS in the docetaxel arm alone (P=0.87), but an association (P0.001) with increased PFS in the vaccine combination arm. [] We have now developed the capability, in collaboration with an NCI core facility, to conduct digital immunohistochemistry (IHC) analyses of biopsy specimens. In a trial of i.t./s.c. vaccination with PROSTVAC, there were statistical increases in CD4+ and CD8+ T cells, and decreases in Tregs in post- vs. pre-treatment biopsies (P0.001 for each). There was an association (P=0.002) in best decrease in serum PSA levels and increases in CD8+ TILs post-vaccination. Immunotherapy trials have been initiated or are planned in prostate, bladder, and lung carcinomas in which pre- vs. post-treatment biopsies will be analyzed by digital IHC and compared with changes in specific immune cell subsets in the periphery.